The present invention relates to mixing apparatus for mixing an incoming fluid stream with a material to be mixed with the incoming fluid stream. More particularly, the present invention relates to mixing apparatus specially adapted for reconstituting powdered cell culture media in predetermined unit volume amounts.
Viable animal cells and tissue in in vitro cultures have been known since the early 1900s. While animal cell culture today is a sophisticated technology, the basic culture technique has not changed since the beginning of the century. Cells or tissue, either primary or transformed, are grown in a liquid nutrient mixture generally referred to as "media." This media is a complex mixture of amino acids, vitamins, salts, and other components. It is often supplemented with 1-10% purified bovine fetal or newborn calf serum. Cell culture media and serum are available commercially from many sources.
While the basic cell culture technique has not changed appreciably over the years, the volume of cell culture and the accessibility of this laboratory technique has increased dramatically. Not only are more research laboratories, pharmaceutical and biotechnology companies employing tissue culture techniques but they are doing so, often, on a relatively large scale. A medical product related corporation may consume tens or hundreds of liters of liquid media a day and employ large numbers of laboratory technicians and scientists to generate antibodies, growth factors or purified protein from tissue culture for commercial use. Thus, between media supply costs and employee time there is a considerable expense associated with the tissue culture process today.
Cell culture media is available commercially either as a dry powder which is reconstituted by adding an appropriate volume of water, or as a pre-packaged liquid. There are also a number of additives that are typically added to the media before use. These include sodium bicarbonate, glutamine, additional buffers or antibiotics.
Pre-packaged liquid is sterile, aliquoted into convenient sizes and is ready to use. However, the media is typically light sensitive and has a prescribed shelf-life. Therefore, media must be ordered on a regular basis. It also should by stored under refrigeration and, in its prepackaged form, requires significant man-power time to unpackage and transport. Further, shipping costs of prepackaged liquid is becoming increasingly more expensive.
Powdered media is provided in bulk or in premeasured packages. It tends to have a longer shelf life, is less expensive and requires less storage space and handling time than the liquid form. However, the powdered media must be dissolved and aliquoted under sterile conditions. The increased handling and preparation time especially for large volume media preparation often makes pre-packaged liquid media the preferred choice despite the increased cost. Thus a powdered media that is easy to prepare, requires less storage space than liquid media and whose preparation requires minimal effort will be a significant improvement over the current art.
Reconstitution of powdered media is a several step process. To prepare a liquid media from a solid powder, a known amount of powder intended for a specific volume of media is measured out and added to a volume of distilled water which is typically slightly less than the final desired volume. The powder and water are stirred until the solid is completely dissolved. Then, a specific quantity of sodium bicarbonate is added and dissolved. Sodium bicarbonate and the powdered media must not be simultaneously added to the water, or a calcium carbonate precipitate forms. The pH may thereafter be adjusted using acid or base and additional water is added to increase the media to its final volume. The entire mixture is then passed through a sterilizing filter. The media may thereafter be collected in a single large sterile vessel, or proportioned into several smaller sterile vessels.
Powdered tissue culture media has a very fine particle size and is hygroscopic. When mixed with water, it tends to "ball" or "clump." Thus, when reconstituting in water, sufficient agitation is required to break up any clumps that may form upon initial contact with water. For smaller batch sizes, sterile magnetic stir bars can be added to the mixing container and the container is then placed on a magnetic stir plate. Additional manipulations are required to add stir bars to the mixing containers. In a typical laboratory setting, magnetic stir plates are not a practical solution for large volume media preparation.
In addition, due to its hygroscopic nature, the media absorbs water when stored, especially in humid environments. Wet media has a shortened shelf-life, becomes lumpy and requires aggressive agitation to reconstitute. Thus, powdered media shelf life could be improved if it were provided in premeasured sealed and desiccated aliquots.
The reconstitution process requires several steps and several separate pieces of equipment. It generally requires at least one vessel, large enough to contain the entire final volume of reconstituted media, plus one or more vessels to receive the sterile media after filtration. The sterilized media is usually delivered into open top containers. Thus, most media preparation is done in a laminar flow hood. Processing large volumes of media in a hood is difficult because there is often not enough space to accommodate the containers and sterile media. A device that would permit the preparation of such a product with minimal physical contact and facilitate media preparation without the inconveniences described above would fulfill a long felt need in the scientific community.
There are a wide variety of solutions, the preparation of which requires the sequential dissolution or addition of components with minimum physical contact. In the research laboratory there are a range of chemicals that are purchased as a powder or series of powders or as a series of concentrates and must be prepared prior to use. Other substances may be toxic so handling should be minimized. Some chemicals are required to be free of nucleases such as those found on human hands and require sterilization before use. Still others must be free from contaminants including dusts, bacteria, viruses and fungi. As a liquid these substances may have a predetermined shelf-life and while they may be inexpensive to purchase as a powder, they are considerably more expensive to purchase and receive in a prepackaged, filtered sterile liquid form.